Marker for colorectal carcinoma and methods of detecting the same

ABSTRACT

A new marker for colorectal carcinoma has been discovered which is a glycoprotein having a molecular weight of approximately 160,000 daltons. Assay methods which can identify this marker are useful indetecting, diagnosing, and monitoring colorectal carcinoma, and in particular, carcinoma of the undifferentiated variety which heretofor were not readily detectible. For example, an assay which utilizes an antibody reacting to this glycoprotein marker is useful in a screening method for the detection and monitoring of colorectal carcinoma cells. Such an antibody can be included as part of a kit for screening a patient for colorectal carcinoma.

BACKGROUND OF THE INVENTION

The U.S. Government has rights in this invention pursuant to NationalCancer Institute Grant Nos. CA 44583 and CA 44704.

CROSS-REFERENCE TO RELATED APPLICATION

The subject matter of this application is related to that of copendingapplication Ser. No. 183,831, assigned to the assignees of thisapplication, entitled "Monoclonal Antibody for Colorectal Carcinoma",filed on even date herewith.

The present invention relates to the field of cancer diagnosis, and morespecifically to the identification of new cancer markers and methodsuseful in the diagnosis, detection, and monitoring of human colorectalcarcinoma.

Colorectal carcinoma is a cancer which affects many people per year. Theprognosis is poor in about 50% of the cases because the tumor is oftennot detected until the disease has spread and has reached a terminalstage. Early diagnosis is important to increase chances of arresting thecarcinoma before it metastasizes, thereby leading to an improvedprognosis.

A widely used method of the identification of cancerous tissue is todetermine its structural resemblance to fetal or immature tissue. Inthis way, tumors can be classified depending on the degree of cellulardifferentiation; they can be undifferentiated, poorly differentiated,moderately differentiated or well differentiated. In addition, thebehavior of a given tumor can often be related to its degree ofdifferentiation. For example, poorly differentiated tumors tend to growmore rapidly and metastasize earlier than do differentiated tumors whichmore closely resemble the tissue of origin. Poorly differentiated tumorstend to have a poor prognosis and are difficult to detect.

One method of early tumor diagnosis is detection of the presence of amarker or antigen specific for a particular tumor. These normallyproteinaceous markers are synthesized by the tumor, and may be found inthe serum and/or on the surface of the tumor. Only a limited number oftumor markers for colorectal carcinoma have thus far been found to haveclinical use. These include carcinoembryonic antigen (CEA), and thesialyated Lewis a antigen (CA 19.9). Unfortunately, approximately 40% ofpatients whose condition has been accurately diagnosed as colorectalcarcinoma do not have elevated plasma levels of either of these antigenswhen initially examined. There is no commercially availableserodiagnostic marker which can be used to detect the tumor and tomonitor therapy for this group.

Production of some tumor markers e.g., CEA and CA 19.9, by tumor cellsin vitro correlates with a greater degree of cellular differentiation.For example, CEA and CA 19.9 are present to a far lesser degree onpoorly differentiated or undifferentiated cancer cells than on thosewhich are more differentiated. Accordingly, many patients withundifferentiated colorectal carcinomas never develop elevated serumlevels of either of these markers, even in the terminal stages of thecancer. There is also considerable overlap between the presence of CA19.9 and CEA, the patient with a normal CEA level and an elevated levelof CA 19.9 being the exception rather than the rule. Therefore, newmarkers suitable for identifying and monitoring undifferentiated tumorswould be of great value.

Accordingly, it is an object of this invention to provide a new markerfor the detection of colorectal carcinoma.

It is another object of the invention to provide a new marker suitablefor diagnosing and monitoring colorectal carcinoma, and in particular,undifferentiated or poorly differentiated colorectal carcinoma for whichknown characterizing markers are not present.

A further object of the present invention is to provide a method and kitfor the detection and monitoring of colorectal carcinoma in patientsusing assay methods specific for markers on colorectal carcinoma cells.

A still further object is to provide screening procedures for detectingthe presence of colorectal carcinoma cells at all stages ofdifferentiation.

SUMMARY OF THE INVENTION

A new tumor marker for human colorectal carcinoma (hereinafter referredto as the CC glycoprotein) has been discovered with the use of anantibody raised to an undifferentiated tumor cell line. This marker is aglycoprotein of approximately 160,000 daltons molecular weight, and isfound on the surface of undifferentiated as well as more differentiatedcolorectal carcinoma cells.

A method for detecting and monitoring human colorectal carcinoma hasbeen developed. This method includes contacting a biological sample withan antibody or portion thereof which reacts with the CC glycoprotein, ananalog, or portion thereof, and observing if the antibody reacts withthe sample. The biological sample may be whole blood, serum, ascitesfluid, a tissue biopsy, a tumor, a tissue culture, or a histologicalpreparation thereof. The antibody may be raised to undifferentiatedtumor cells, to the CC glycoprotein, or to analogs or portions thereof.More specifically, this antibody may be a polyclonal or monoclonalantibody, or analog or portion thereof which does not cross-react withCEA, NCA, CA 19.9, alpha-1-acid glycoprotein, or with the blood groupsubstances A, B, and H. An immunoassay may be utilized to observe theextent of reaction between the receptor and the sample.

A detection method has also been developed which enables the diagnosisand identification of a tumor cell in a biological sample from apatient. In this method the sample is subjected to at least one of aplurality of tests, each of which is specific for a particular tumormarker. The test may be any type of assay, preferably an immunoassaywhich employs an antibody specific for a tumor marker. The tests may becarried out sequentially until one of them indicates the presence of aparticular marker.

Further, a kit for screening a patient for colorectal carcinoma has beendeveloped which contains a plurality of different antibodies specificfor tumor markers. These antibodies may include those reacting with oneof the following markers: CC glycoprotein; CEA; NCA; CA 19.9; andalpha-1-acid glycoprotein.

The foregoing and other objects of the present invention, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description of the invention.

DESCRIPTION

The present invention relates to a marker and method for detecting humancolorectal carcinoma. More particularly, it relates to the use of assaymethods which enable the detection of the marker. Detection of themarker is indicative of the presence of colorectal carcinomas, such asthose which are undifferentiated, which otherwise would not have beendetected with antibodies to other tumor markers.

CC glycoprotein, the newly discovered marker has been found on thesurface of many tumor cell lines including those which elicit CEA andsome which do not. The following procedure describes its isolation fromthe undifferentiated, non-CEA-producing cell line MIP 101. However,other undifferentiated cell lines could be employed as the startingmaterial as well.

Radiolabeled, undifferentiated MIP 101 tumor cells are lysed with adetergent-containing buffer. The lysate is then subjected toimmunoprecipitation procedures using an antibody which reacts with theCC glycoprotein. Polypeptides in the immunoprecipitates are thenseparated by electrophoresis on polyacrylamide gels containing sodiumdodecyl sulfate (SDS), and identified by autoradiography. The marker hasthus been identified as the 160,000 dalton CC glycoprotein.

The antibody useful in detecting the marker may be a polyclonal antibodyor portion thereof raised to undifferentiated tumor cells, and shown toreact with the CC glycoprotein, analogs, and fragments thereof. However,the antibody is most preferably a monoclonal antibody or fractionthereof. It does not cross-react with other known tumor markers such asCEA, CA 19.9, NCA, and alpha-1-acid glycoprotein, or with any of theblood group substances A, B, or H, as determined by established ELISAprocedures.

This antibody can be of any class and subclass, but the ND4 monoclonalimmunoglobulin produced by the forementioned hybridoma was determined tobe of the IgG2a subclass as by the Ouchterlony double diffusion test.This antibody may also be synthesized by biosynthetic or recombinantmeans, either in whole or in part, and may be linked to other functionalmolecules such as toxins, dyes, enzymes, or radioactive markers.

The antibody used to detect the marker is a monoclonal antibody calledND4; however, other antibodies which react with the marker are useful aswell. The ND4 antibody was obtained from a hybridoma cell line formedupon the fusion of a mouse myeloma cell with a spleen cell from a mousewhich had been previously immunized with undifferentiated ornondifferentiated tumor cells. Preferably, non-CEA and/or non-CA 19.9-producing and undifferentiated colorectal cancer cells such as MIP 101cells are used as the immunizing agent. However, the immunogenalternatively may be the CC glycoprotein, analogs or portions thereof.The mice whose spleen cells were chosen for fusion are preferably from agenetically defined lineage such as Ba1b/c. The myeloma cells used inthe fusion are from a mammalian, antibody-producing cell line, but mostpreferably are from a mouse cell line, an example being NS-1. Themonoclonal antibody can be obtained from ascites fluid of mice injectedwith the forementioned hybridoma.

The antibody is specific for tumor cells and many fetal tissues, asdetermined by fluorescence binding studies performed on formalin-fixedcells, and will recognize tumor cells having different degrees ofdifferentiation including some CEA- and/or CA 19.9-producing cells.However, this antibody is particularly useful in recognizing tumor cellswhich are undifferentiated or poorly differentiated, and which do notelicit detectable levels of CEA and/or CA 19.9.

Alternatively, the antibody may be a polyclonal immunoglobulin obtainedfrom the serum of an animal previously immunized with the CCglycoprotein.

The present invention further provides a method for detecting tumorcells, which includes contacting a biological sample with a receptorwhich is capable of reacting with the CC glycoprotein, analogs, andfragments thereof, and observing whether the receptor reacts with thesample.

Examples of biological samples which may be examined by this methodinclude tissue biopsies, tumors, blood, serum, ascites fluid, tissuecultures, and histological preparations thereof. A histologicalpreparation of a biological preparation may include a wet mount, a drymount, a frozen sample, a paraffin-embedded sample, an acrylic-embeddedsample, or a sample adapted in any way for microscopic examination.

A reaction between the antibody and a sample may be detected by an assayincluding, for example, one which is enzyme-linked and one which isimmunological. A preferred assay is an immunoassay such as aradioimmunoassay, Western blot, or a nitrocellulose "dot" blot.

The method of the present invention can be employed for diagnostic andprognostic purposes, for example, to monitor the growth of a tumor orthe functional status of normal colorectal cells. More specifically, themethod can be employed, for example, to diagnose the in vivo presence oftumor cells which are undifferentiated or poorly differentiated, andwhich do not produce CEA and/or CA 19.9.

This screening method can also be expanded to include the use ofmonoclonal antibodies which are specific for other tumor markers, suchas CEA and CA 19.9, so as to enable the determination of the level ofdifferentiation which a tumor cell has achieved. The presence of CEA orCA 19.9 has been correlated with higher degrees of differentiation whilethe presence of the CC glycoprotein has been correlated with both highand low degrees of differentiation. Therefore, a tumor cell which testspositively for the CC glycoprotein, but tests negatively for CA 19.9 andCEA is most likely undifferentiated or poorly differentiated.

The instant invention also relates to a method for screening patientsfor colorectal carcinoma. It includes subjecting a biological sample (aspreviously defined) to at least one test selected from a plurality oftests, each of which is specific for a colorectal carcinoma cell marker,and correlating the presence of a specific marker with a degree ofdifferentiation of that cell.

The screening method of the present invention includes tests for tumormarkers CEA, CA 19.9, NCA, alpha-1-acid glycoprotein, and the CCglycoprotein, as well as any additional markers which indicate thepresence of colorectal carcinoma. The tests performed may be assays, forexample, to determine enzyme-linked activity, or may be immunoassayswhich utilize an antibody specific for a particular marker. They may beperformed in a sequential manner until the presence of at least onemarker has been proven.

Finally, this invention provides a convenient kit for screeningbiological samples for colorectal carcinoma. This kit includes antiseraor antibodies specific for tumor markers such as the CC glycoprotein,NCA, CA 19.9, CEA, alpha-1-acid glycoprotein, and may also include anyother relevant antisera or antibodies. Screening may be performed by anyimmunoassay procedures such as, for example, radioimmunoassay, Westernblot analysis, or nitrocellulose "dot" analysis.

The following examples illustrate the best mode of making and practicingthe present invention, but are not meant to limit the scope of theinvention, since alternative methods may be used to obtain similarresults.

EXAMPLE 1 Hybridoma and Monoclonal Antibody Production

Ba1b/c mice (The Jackson Laboratory, Bar Harbor, Me.; 6-8 weeks old )were immunized with four injections of MIP 101 cells, a colorectalcancer cell line of undifferentiated morphology (Niles, et al., CancerInvest., in press (Dec., 1987)). The injections were performed one weekapart and 5×10⁶ cells were injected on each occasion. The first threeinjections were given intraperitoneally, and the fourth intravenously.Cells were injected with complete Freunds adjuvant on the firstoccasion, incomplete adjuvant on the second and third occasions, andwithout adjuvant on the last occasion. Serum withdrawn prior to the lastinjection showed prominent binding to both the immunogen (MIP 101 cells)and another poorly differentiated cell line, Clone A (derived from DLD-1(ATCC No. CCL221) by D. Dexter, Roger Williams Hosp., Providence, R.I.,now at Dupont, Wilmington, Del.) using a solid phase microtiter plateenzyme-linked immunoassay. The mouse with the best immune response wassacrificed three days after the last injection.

Hybridomas were produced by fusion of spleen cells from the sacrificedmouse with NS-1 (P3NS-1/1-Ag4-1) myeloma cells (American Type CultureCollection, Rockville, Md.; Acc. No. TIB18). In the present example, themethod of Nadakavukaren (Differentiation 27: 209-202, (1984)) wasemployed to perform the fusions. Resultant clones were tested forbinding to MIP 101 cellas and to Clone A. Subcloning by serial dilutionwas carried out on one clone. The most productive subclone was injectedinto the peritoneal cavity of Ba1b/c mice to produce ascites fluidcontaining monoclonal antibody. The hybridoma which produces thissubclone was deposited with the American Type Culture Collection(Rockville, Md.; ATCC No. HB 9600) on 12/8/87. The ascites fluidobtained was centrifuged, tested for activity, and then stored at -70°C. until required. This source all subsequent investigations.

EXAMPLE 2 Characterization of Resulting Monoclonal Antibodies A. Bindingof Monoclonal Antibodies to Cell Lines In Vitro.

Binding studies were performed on formalin-fixed cells using the ND4antibody and rhodamine conjugated rabbit anti-mouse antiserum. In thepresent example, the method of Bleday et al. (Cancer 57: 443-440,(1986)) was followed. Cells were viewed to determine binding using aZeiss epiflourescent microscope. Twenty eight different cell lines wereexamined: fifteen human colorectal lines, including nine which producedCEA and six which did not (Table 1); seven human, non-colorectalcarcinoma lines (Table 2); and six normal, non-human cell lines (Table2). Ascites fluid from an IgG2a-producing mouse myeloma line UPC-10(Sigma) was used as a negative control antibody. Binding to cell lineswas graded "-," "+" or "++" dependant on degree of fluorescence. Celllines were obtained from several sources. All CCL lines, MCF-7, CRL1420, CV-1, and A431 were obtained from the American Type CultureCollection. CX-1 was obtained from S. Bernal (Dana-Faber CancerInstitute, Boston, Mass.); Moser was from M. Brattain (Baylor College ofMedicine, Houston, Tex.); and EJ, RT112 and MB49 were from I.Summerhayes (New England Deaconess Hospital, Boston, Mass.). DLD-2 andDLD-1-derived Clones A and D were obtained from D. Dexter (RogerWilliams Hospital, Providence, R.I., now at Dupont, Wilmington, Del.).

                  TABLE 1                                                         ______________________________________                                        HUMAN        CEA          ND4                                                 COLORECTAL   PRODUCTION   IMMUNO-                                             CARCINOMA LINE                                                                             (ng/10.sup.6 cells/ml)                                                                     FLUORESCENCE                                        ______________________________________                                        CCL 235      523          +                                                   CCL 238      362          ++                                                  DLD 2 120    +                                                                CX1 70       ++                                                               CCL 222      2.5          ++                                                  CCL 228      0.7          -                                                   CCL 231      0.6          ++                                                  CCL 227      0.15         ++                                                  MOSER (+)    ++                                                               CCL 220      0.0          ++                                                  CCL 220.1    0.0          ++                                                  CCL 224      0.0          ++                                                  MIP 101      0.0          ++                                                  CLONE A      0.0          ++                                                  CLONE D      0.0          ++                                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                 ND4              IMMUNO-                                             CELL LINE                                                                              TISSUE SOURCE    FLUORESCENCE                                        ______________________________________                                        CRL 1420 Human Pancreatic Cancer                                                                        -                                                   MCF7     Human Breast Cancer                                                                            ++                                                  EJ       Human Bladder Cancer                                                                           -                                                   RT 112   Human Bladder Cancer                                                                           -                                                   A431     Human Vulval Cancer                                                                            -                                                   CCL 105  Human Adrenal Cancer                                                                           +                                                   CCL 185  Human Lung Cancer                                                                              -                                                   LOX      Human Melanoma   -                                                   KAPOSI   Human Sarcoma    -                                                   CV-1     Normal Monkey Kidney                                                                           -                                                   MB49     Normal Mouse Bladder                                                                           +                                                   THE      Normal Hamster Stomach                                                                         -                                                   F111     Normal Rat Fibroblast                                                                          -                                                   A31      Normal Mouse Fibroblast                                                                        -                                                   CCL 22   Normal Bovine Kidney                                                                           -                                                   ______________________________________                                    

After formalin fixation, indirect immuofluorescence showedrhodamine-conjugated antibody binding to 14 out of 15 human colorectalcarcinoma cell lines, with prominent (++) binding to 12 out of 15 (80%)lines. Prominent binding was also seen on all of the 6 non-CEA-producingcolorectal carcinoma cell lines. Two of seven human, non-colorectalcarcinoma lines and one of six non-human lines also bound the antibody.

B. Immunohistopathology.

Immunohistopathological studies were performed to determine whether theND4 antibody binds to a cancerous or normal human tissue and if so, onwhich cells in the tissue section.

Formalin-fixed, paraffin-embedded tissue sections were deparaffinizedwith xylene and ethanol, rehydrated, and incubated with phosphatebuffered saline (PBS) containing 0.3% H₂ O₂ to block endogenousperoxidase activity. Sections were washed and blocked with 1% horseserum for 30 minutes and then incubated at 4° C. overnight with ND4antibody. Subsequent staining was performed according to a standardavidin-biotin-immunoperoxidase technique. In the present example, themethod of Hsu et al. ((1981) Cytochem. 29:1349-1353) was followed.Tissue specimens were obtained from the Department of Surgery, DeaconessHospital, Boston, Mass. or the Department of Pathology, Boston CityHospital, 0554 Boston, Mass.

The following tissues were examined: colon carcinoma, including two welldifferentiated, nine moderately differentiated, and four poorlydifferentiated or undifferentiated tumors; normal tissue from thirteendifferent specimens of normal colonic mucosa obtained from patients witheither benign colonic diseases or entirely normal colons, or from themargin of colonic resections from patients who had undergone surgery forcolorectal cancer; twenty one different normal non-colonic tissuesamples obtained by autopsy; and samples from eleven non-colonic tumors,of which two were gastrointestinal in origin. The results are shown inTable 3.

                  TABLE 3                                                         ______________________________________                                        BINDING OF ND4 MONOCLONAL ANTIBODY TO                                         COLORECTAL CARCINOMA.                                                                        NO.         % OF SAMPLES                                       TISSUE         of SAMPLES  BINDING                                            ______________________________________                                        Colorectal carcinomas                                                                        15          60%                                                (including 3 of 4 poorly                                                      differentiated tumors)                                                        Normal colonic epithelium                                                                    13          15%                                                Normal non-colonic tissue                                                                    21           5%                                                Non-colonic tumors                                                                           11           9%                                                ______________________________________                                    

B. Antibody Typing

ND4 antibody was screened for antibody isotype by the Ouchterlony doublediffusion test in agar plates against anti IgM, anti IgG, anti IgG1,anti IgG2a, anti IgG2b and anti IgG3 antibodies (Cappell). Precipitatingbands were produced only with anti-IgG2a antibodies.

EXAMPLE 3 CC Glycoprotein Isolation

Antigen characterization was performed by immunoprecipitation of MIP 101cells which had been radiolabelled by three different procedures. Cellswere labelled overnight with [³⁵ S]-methionine, or with [³H]-glucosamine (New England Nuclear) according to the method ofKurzinger et al. (J.B.C. 257: 20,12412-12418, (1982)). Cell surfaces oflive MIP 101 cells were labelled with ¹²⁵ Iodine using thelactoperoxidase procedure of Morrison (Methods Enzymol. 70: 214-220(1980)). The labelled cells were lyzed in RIPA buffer containing 150 mMsodium chloride, 1% Triton-X-100 (Sigma), 0.1% sodium dodecyl sulfate(SDS; Biorad) and 10 mM Tris-HCl pH 7.2, to which 1 mMphenylmethylsulphonyl fluoride (Sigma) was added. Immunoprecipitationswere carried out using Protein A-Sepharose (Sigma) to which agoat-anti-mouse whole serum immunoglobulin (Cappell) was linked. Thecell lysate was preincubated at 4° C. with the Protein A complex.Immunoprecipitations with ND4 antibody were carried out as previouslydescribed (Hsu et al. Cytochem. 29: 1349-1353) 1981)). Precipitationswith UPC-10 (Sigma) or without antibody were used as negative controls.Immunoprecipitates were analyzed by SDS-polyacrylamide gelelectrophoresis on 10% gels, followed by autoradiography of the driedgels with Kodak X-OMat film. For the [³⁵ S]-methionine and [³H]-glucosamine-labelled cells, the gels were soaked in EnHance (Dupont)for 45 minutes, and were then washed in distilled water for 30 minutesprior to drying and exposure to X-ray film.

Immunoprecipitation of antigen labelled with ¹²⁵ Iodine, [³⁵S]-methionine and [3H]-glucosamine each showed a band of approximately160,000 daltons molecular weight on SDS-polyacrylamide gels. This bandwas absent when either control antibody or no antibody was used.

EXAMPLE 4 Characterization of CC Glycoprotein A. Effect of Trypsin onND4 Antibody Binding.

To determine if the marker recognized by the ND4 antibody were aprotein, MIP 101 cells which were known to have this marker werepretreated with the serine protease, trypsin, to determine ifrecognition would be affected. Ninety six well, sterile ELISA microtiterplates were seeded with MIP 101 cells which were grown until confluent.Cells were then fixed with 40% formaldehyde at 24° for 15 minutes, andthen rinsed twice in PBS. Two lanes of cells were incubated with 1%trypsin (Gibco) at 37° for two hours and two lanes were incubated withPBS as positive controls. All lanes were examined by light microscopy toensure that cells had not detached from the microtiter plate.Trypsin-treated and PBS-treated wells were incubated for one hour at 37°C. with ND4 antibody in serial dilutions from 1:100 to 1:100,000. Theplates were washed and then incubated with peroxidase-conjugatedgoat-anti-mouse immunoglobulin (Hyclone). After further washes theplates were developed using orthophenyline diamine as a substrate. Theplates were read in a Biorad EIA reader at an absorbance of 492 nm.Optical density was plotted against ND4 antibody dilution for bothtrypsin-treated and PBS-treated lanes.

Trypsin treatment of formalin-fixed MIP 101 cells resulted in lossof >90% of the binding of ND4 antibody to the cells, as determined bythe ELISA, indicating that the antigen recognized by the ND4 antibody ispresent as a polypeptide residing on the surface of MIP 101 cells.

B. Cross-Reactivity with Other Antigens.

Purified CEA, NCA, and alpha-1-acid glycoprotein as well as blood groupantigens obtained from saliva of patients of AB and O blood types werebound to wells of ELISA microtiter plates using 0.1 bicarbonate buffer,pH 9.6. After blocking with 3% bovine serum albumen in Tris-bufferedsaline for one hour at 37° C., the wells were incubated with ND4antibody and antibodies to blood group substances A, B, and H aspositive controls for the saliva-derived antigens. Subsequent reactionswere carried out as described for the ELISA procedure mentioned above

No cross-reactivity of the ND4 antibody with CEA, NCA, or alpha-1-acidglycoprotein, or with any of the blood group antigens A, B and H wasdetected by this test.

EXAMPLE 5 Antigen Detection in Patients with Colorectal Cancer

Ascites fluid from two patients, serum from 37 patients with colorectalcancer and serum from 11 normal subjects were assayed for the presenceof the CC glycoprotein antigen using a nitrocellulose "dot" immunoassay.Nine of the patients had early stage disease. Five microtiter "dots" of1:5 dilutions of ND4 antibody-containing ascites fluid or serum in PBSwere placed on a nitrocellulose membrane. After allowing these dots toair dry, the membrane was blocked with 3% bovine serum albumen for onehour at 20° C. The membrane was incubated with a 1:100 dilution ofascites fluid at 4° C. overnight, washed with PBS, and incubated withsheep-anti-mouse IgG that had been preabsorbed with a mixture ofpolymerized whole serum and human IgG (Cappell) for two hours at 20° C.The reaction was developed with 4-chloro-2-napthol and H₂ O₂.

Nitrocellulose dot immunoassays of serum from patients with colorectalcancer showed positive reactions in 15 of 37 patients (41%). Threepatients had early stage disease and had CEA levels of less than 2.5ng/ml. Three patients with positive assays prior to resection of theirprimary tumor had negative reactions postoperatively.

As illustrated by the forementioned examples, a hybridoma-derivedmonoclonal antibody which reacts with a newly discovered tumor marker,CC glycoprotein has been produced, characterized, and successfullyemployed to detect colorectal carcinomas which may otherwise would nothave been detected with antisera specific for other tumor markers.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the present inventionbeing indicated by the appended claims rather than by the foregoingdescription, and all the changes which come within the meaning and rangeof equivalency of the claims are therefore intended to be embracedtherein.

We claim:
 1. A method of detecting human colorectal carcinoma comprisingthe steps of:(a) contacting a biological sample with an antibody orportion thereof which reacts with CC glycoprotein, analogs, or fragmentsthereof, CC glycoprotein having a molecular weight of approximately160,000 daltons, and not cross-reacting with antibodies to any ofcarcinoembryonic antigen, CA 19.9, alpha-1-acid glycoprotein,nonspecific cross-reacting antigen, or to any of blood group substancesA, B, or H; and (b) observing whether said antibody reacts with saidsample, an observation of a reaction with said sample indicating thepresence of human colorectal carcinoma.
 2. The method of claim 1 whereinsaid antibody comprises a monoclonal antibody, analog, or fragmentthereof capable of reacting with CC glycoprotein.
 3. The method of claim1 wherein said sample comprises biological material selected from agroup consisting of tumors, whole blood, serum, ascites fluid, andtissue biopsies, tissue cultures, and in vitro histological preparationsthereof.
 4. A kit for screening a patient for colorectal carcinoma, saidkit comprising antisera specific for at least one marker for colorectalcarcinoma, said marker including the CC glycoprotein, and said screeningis carried out by subjecting a tissue sample from said patient toimmunoassay procedures utilizing said antisera.